1. Field of the Invention
The present invention relates to the irreversible immobilization of enzymes into polyurethane coatings.
2. State of the Art
Immobilization has been widely employed to enable and enlarge the application of enzymes as catalysts in industrial processes. Polyurethane foam has been employed as polymeric support for bioplastic synthesis with various enzymes over the last decade. Polyurethane sponge-like polymers may be synthesized from hydrophilic toluene di-isocyanate (TDI)- or methylene bis (ρ-phenylisocyanate) (MDI)-based polyisocyanate prepolymers and water. The incorporation of enzymes in monolithic polyurethane foam is often characterized by a degree of immobilization close to 100% and a high activity retention. Thermostability enhancement via immobilization in polyurethane foams has also been reported.
The insertion of biological molecules in coatings and thin films may drive a large range of applications. For example, potentiometric biosensors often involve the covalent attachment of enzyme onto an inner film adjacent to the sensing surface of the electrode, and the subsequent protection of the enzyme layer with an outer film. Another immobilization method for the fabrication of amperometric biosensors relies on the entrapment of enzyme in a gel layer, which is further coated by an external protective film. The lifetime and use of such systems are often limited by the diffusion of enzyme through the external membrane. To overcome this main disadvantage, the enzyme has to be directly and covalently immobilized into the coating. The covalent incorporation of biocatalyst into coatings would also be beneficial for other bioprocesses such as biocatalytic separation and filtration, microchips, and antifouling.
Direct covalent immobilization of highly-active enzymes into coatings and films has remained an elusive goal, with some of the most successful approaches exhibiting only up to 0.5% activity. Waterborne polyurethane coatings result from the polymerization of aqueous polyester based polyol dispersible aliphatic polyisocyanates. As the film is cured at room temperature, water evaporates and cross-linking occurs through the condensation between hydroxyl groups and isocyanate functionalities. Two-component waterborne polyurethanes are increasingly used in industrial applications, as they exhibit properties similar to those of solvent borne polyurethane coatings. Waterborne polyurethane coating represents a potentially ideal polymeric matrix for multipoint and covalent immobilization of enzymes.
In view of the foregoing, there is a need in the art for a method by which an enzyme can be directly added to the aqueous phase of a two-component system prior to polymerization. The immobilization process relies on the ability of amines at the enzyme surface to react with isocyanate functionalities at a faster rate than hydroxyl groups on the prepolymer.